The autoimmune lymphoproliferative syndrome (ALPS) is a human disorder due to defective lymphocyte apoptosis resulting in abnormalities in lymphocyte homeostasis. This produces a combination of lymphadenopathy, autoimmunity and increased risk of lymphoma. The diagnostic criteria used to diagnose ALPS include a triad of findings: lymphadenopathy, increased circulating alpha-beta double negative T cells and defective in vitro Fas mediated lymphocyte apoptosis. The majority of patients with ALPS have been found to have a heterozygous mutation in the gene encoding Fas (CD95) while a small number of patients have been found to have mutations in Fas ligand or caspase 10. However, a sizeable number of ALPS patients do not have mutations in the genes noted about and are categorized as having ALPS type 3 with an uncharacterized defect. We have begun a systematic evaluation of ALPS type 3 patients by first collecting all the various laboratory data that has been identified as abnormal in ALPS type 1a to see if these findings differ in ALPS type 3 patients. In addition, we have begun a systenatic evaluation of intracellular proteins involved with the apoptotic process. These are targeted at assessing the assemblage of the Death Inducing Signalling Complex (DISC) and downstream events and proteins that control the apoptotic cascade that ultimately results in cell death. We are also attempting to identify ALPS type 3 patients who have relatives with similar clinical findings in an attempt to identify genetic links to disease development but to date all of our cases are isolated. We have developed a very complete ALPS database that includes all ALPS type 3 patients seen at the NIH. One issue that has emerged is that a number of the ALPS type 3/ALPS phenotype patients actually represent ALPS type 1a patients with somatic mutations in Fas because the level of cells containing the defective gene falls below 20% and hence is not detected using conventional genomic sequencing and the only cells with an apototic defect at those containing the mutant gene and they represent a minority of the lymphocytes. Consequently we are now reviewing all ALPS type 3 and ALPS phenotype patients for the possiblity that some have a somatic Fas mutation. To date we have identified 11 such patients. Because of this finding, we are using the extensive clinical and laboratory database that we have created to determine if there are biomarkers that will reliably distinguish those patients who are most likely to have a somatic Fas mutation. At this juncture it appears that having double negative T cells above 4% together with a soluble FasL level (or an elevated vitamin B12 or an elevated IL-10) level will differentiate between those with a muation and those without a definable mutation. Finally, we hav begun expression microarray evaluation of ALPS type 3 patients focusing intitially on non-activated T cells with plans to also evaluate activated T cells compared to control T cells.